Neutrophil priming and activation following trauma and sepsis is a key event implicated in causing Adult Respiratory Distress Syndrome (ARDS), Acute Lung Injury (ALI) and Multi-Organ Failure Syndrome (MOSF). Priming of the respiratory burst by cytokines following injury and sepsis results in excessive superoxide production by the NADPH oxidase leading to auto-inflammatory tissue damage. Many of the molecular mechanisms involved in priming and activation of the NADPH oxidase, however, remain poorly defined. Our long-term goal is to develop a detailed molecular understanding of how protein kinase and lipid kinase signaling pathways, including the PI 3-kinase pathway, the p38MAPK pathway and the Erk1/2 pathways regulate the assembly, subcellular targeting, and activity of the neutrophil NADPH oxidase during priming and activation. Our previous work and preliminary observations identified PX domains in the p47phox and p40phox subunits as modular protein domains that bind to specific lipid products of PI 3-kinase, and showed that the priming agents PAF and TNF1 induced the assembly of a p47phox:p67phox:p40phox heterotrimeric complex in the cytoplasm of primed but un-activated cells. In the studies outlined in this proposal we investigate the importance of PX domain-mediated interactions with lipids and the cytoskeleton, and p47phox binding to PDZ- domain-containing proteins, in neutrophil priming and activation using biochemistry, cell biology and mouse models. In addition, we further explore protein kinase-dependent molecular mechanisms involved in heterotrimer formation during priming. The results from these studies may assist in the development of novel diagnostic or therapeutic reagents aimed at limiting the auto-inflammatory tissue damage patients suffer as a result of sepsis and trauma. PUBLIC HEALTH RELEVANCE3 Neutrophil priming and activation following trauma and sepsis is a key event implicated in causing Adult Respiratory Distress Syndrome (ARDS), Acute Lung Injury (ALI) and Multi-Organ Failure Syndrome (MOSF), which are the leading causes of death in adult surgical intensive care units. Priming of the neutrophil respiratory burst by cytokines following injury and sepsis results in excessive superoxide production by the NADPH oxidase and contributes to the auto-inflammatory tissue damage seen in these syndromes. Our research is designed to better understand regulation of the NADPH oxidase following trauma and sepsis, and facilitate the development of novel diagnostic and therapeutic agents that will reduce tissue damage, morbidity and mortality in these critically ill patients.